Engineered nuclease technology including zinc fingers, TALENs, and CRISPR-Cas9 nucleases, is revolutionizing biomedical research and providing important new modalities for therapy of gene-based diseases. Sensitive detection of off-target effects is important for translating these methods into human therapeutics. In vitro biochemical methods for finding off-targets offer potential advantages of greater reproducibility and scalability while avoiding limitations associated with strategies that require the culture and manipulation of living cells.